ABSTRACT Bioengineering approaches to protein and nucleic acid-based therapeutics require either detailed structure- activity knowledge (e.g. rational design) or robust high-throughput recombinant DNA/protein capabilities (e.g. directed evolution), thus limiting successful implementation. Herein, we propose to investigate ancestral sequence reconstruction (ASR) as a tertiary approach to biopharmaceutical engineering. ASR employs predictive models of molecular evolution with knowledge of extant protein diversity to identify, characterize, and bioengineer desired properties. Coagulation factor VIII (FVIII) represents an attractive target for ASR as current biopharmaceuticals, although efficacious, possess significant pharmacological limitations. To address these limitations, we preliminarily have resurrected ancestral (An) FVIII variants and already identified molecules possessing superior biosynthetic efficiency, specific-activity, and stability. Furthermore, certain An-FVIII constructs display reduced binding and procoagulant inhibition by anti-human FVIII antibodies. ASR also has facilitated the identification and elimination of a previously uncharacterized inhibitory epitope on FVIII through single amino acid bioengineering. In the current application, we propose to integrate ASR, molecular and cellular biology, protein chemistry, and immunobiology to obtain new insights into FVIII that will provide a framework for the future development of improved therapeutics. More broadly, we expect that the proposed studies and subsequent findings will validate and promote ASR as an enabling platform approach towards improving our basic and translational understanding of vertebrate hemostasis and thrombosis.